I am working on the interaction between E. coli DnaK and DnaJ proteins, specifically defining the functional domains of DnaK and DnaJ by mutational and biochemical analysis. I propose to use the Computer Graphics Lab facilities to model the DnaK-DnaJ chaperone interaction. DnaK, a prokaryotic member of the hsp70 family of chaperones that is involved in protein folding exhibits a low intrinsic ATPase which is modulated by its cohorts, DnaJ and GrpE, as well as by substrate proteins. The site of GrpE binding to DnaK has been identified; however, nothing is known about how DnaJ binds DnaK. To this end, we have embarked on a search for DnaK mutants that are allele-specific suppressors of two different dnaJ mutations: dnaJ259 (His33Gln) and dnaJ236 (Asp35Asn). Each of these dnaJ alleles is located in an extremely highly conserved surface loop that is postulated to interact with DnaK. A plasmid-born copy of dnaK under control of the trc promoter was randomly mutagenized with PCR, transformed into the dnaJ mutant strains and selected for cells able to grow at 43C. Among these cells that were able to replicate bacteriophage, some were saved for further characterization. We have identified three dnaK suppressor mutations that suppress both the growth defect and l phage replication defect of the original dnaJ236 mutation. Suppression is allele-specific: none of these mutations suppress either dnaJ259 or =90dnaJ. Each suppressor allele contained multiple mutations, which we dissected by fragment swapping. For dnaK4, a single amino acid change, R167H was necessary and sufficient for suppression. For dnaK43, an amino acid change at I169F exhibited partial suppression, and when combined with T215A, almost full suppression was obtained. For dnaK114, full suppression required the combinatorial effects of multiple mutation. Interestingly, R167H and I169F are located in the highly conserved beta-sheet strand of the amino terminal ATPase domain, suggesting that these amino acids play an important role in interaction with DnaJ. The coordinates of the ATPase domain of a DnaK homologue, bovine hsc70, are available. I propose to use these coordinates to create a model of the interaction mechanism between the ATPase domain of DnaK and DnaJ protein to carry out chaperone function, and to examine the possible effects of our mutation in DnaK on the binding to DnaJ protein. I also will use the graphics facilities to examine the binding affinity of the DnaK-DnaJ interaction by alanine substitutions on DnaK.